PROJECT SUMMARY/ABSTRACT IFN? and IFN? (type I) and IFN? (type II) are potent mediators of anti-cancer immunity having both direct anti- proliferative effects against many cancers as well as a multitude of anti-tumor immunotherapeutic effects. However, while IFN has efficacy against multiple human cancers its clinical utility to date has been limited by the inability to achieve effective concentrations of IFN at tumor sites without causing systemic toxicity. Several groups including ours have successfully approached this problem by using the tumor-targeting ability of monoclonal antibodies to carry IFNs directly to tumor sites. These approaches have shown great promise and anti-CD20-IFN?2 is in clinical trials by ImmunGene and anti-CD138-IFN?2 is being developed by Qwixel Therapeutics under a license from UCLA. Although using the antibody binding specificity to target tumor- associated antigens delivers a greater percentage of the IFN to the site of the tumor than is achieved when IFN is injected by itself, the attached interferon still is recognized and bound by interferon receptors expressed throughout the body that are not tumor associated. This binding can result both in less IFN reaching the tumor and in unwanted off-target toxicity. The goal of the present studies will be to produce anti-IFN fusion proteins in which the IFN will only bind IFN receptors once it reaches the tumor. To achieve that goal we will attach a mask to the IFN, which will inhibit its activity. This mask will be separated from the IFN by a peptide linker that is a site for proteolytic cleavage. A hallmark of cancer is increased proteolytic activity in tumor tissue and the surrounding microenvironment. We therefore hypothesize that we can achieve improved tumor targeting of IFN activity by producing it as a prodrug that will be activated only in the tumor microenvironment following removal of the mask by the tumor associated proteases. Preliminary studies have identified a potential mask and cleavable peptide linker for IFN?. We will now use tumor cell lines and extracts to confirm that the mask inhibits IFN? activity, that it can be removed within the tumor microenvionment and that the unmasked protein is now active both in vitro and in xenograft models. Humanized NSG mice will be used to determine if the masked interferon fusion protein elicits a decreased inflammatory response compared to unmasked IFN. Similar approaches will be used to produce a masked IFN? using either a peptide or scFv mask. The resulting masked IFN? will be characterized as will be done for IFN?. With the successful completion of these experiments, Qwixel will have novel IFN fusion proteins that are even more effective in delivering large amounts of IFN to tumor sites in the absence of systemic toxicity.